This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core 1 focuses on developments associated protein localization within the cell, the protein-protein interactions, the quantitative dynamics and protein trafficking, and the modification states of proteins applying high throughput proteomics measurements. For this annual report advancements in top-down proteomics by Center-investigators are provided;advancement in applying gas phase fractionation with FAIMS are described;measurements of posttranslational modifications including the phosphoproteome with measurements of low abundance pTyr modifications in addition to the more abundant pSer and pThr modifications;and an enhanced version of RePlay proteomics is reported. Advanced methods of sample preparation (e.g., trypsin coated nanoparticles;the biofluids CSF and saliva), pressure digestion and sonication, have been developed in support of measuring differential protein abundances for systems biology studies. Core 1 also includes the research area of activity-based proteomics (ABP and MABPP) that provides a more targeted approach to probing the sub-proteomes of tissues, cells, subcellular fractions, and biofluids. Methods in subcellular fractionation are being implemented in the Center to achieve Core 1 goals for characterizing proteins in cells and their associated localization and trafficking. Additional progress has been made on profiling the cell surface membrane proteome, sperm maturation, oncogenic virus detection, and genome annotation.